Anti ccr7 percp cy5

Manufactured by BioLegend

Sourced in United States

The Anti-CCR7-PerCP/Cy5.5 is a flow cytometry reagent that binds to the CCR7 receptor expressed on T cells and other leukocytes. It is conjugated with the PerCP/Cy5.5 fluorophore, which can be detected using standard flow cytometry equipment.

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3 protocols using anti ccr7 percp cy5

Multicolor Flow Cytometry of PBMCs

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The PBMCs were stained with CMV, GPC3 or HIV Dextramer-RPE (Immudex, Copenhagen, Denmark) for 10 min at room temperature and with anti-CD8-FITC (ProImmune) or anti-CD8-APC (BioLegend, San Diego, CA), anti-CD45RA-FITC (BD Biosciences, San Jose, CA, USA), and anti-CCR7-PerCP/Cy5.5 (BioLegend) for 20 min at 4°C. To detect γδ T cells, PBMCs were stained with anti-TCR-Vγ9-FITC (Beckman Coulter, Erembodegem, Belgium) and anti-CD3-PC5 (BioLegend) for 20 min at 4°C. γδ T cells, with or without zoledronate activation, and TNF-DCs were stained with anti-HLA-class I-FITC, anti-CD80-FITC, anti-CD83-FITC and anti-CD86-PE (BD Biosciences) antibodies for 20 min at 4°C. Flow cytometry analysis was carried out using FACSCanto II (BD Biosciences).

YOSHIKAWA T., TAKAHARA M., TOMIYAMA M., NIEDA M., MAEKAWA R, & NAKATSURA T. (2014). Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy. International Journal of Oncology, 45(5), 1847-1856.

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Dissection and Analysis of T Cell Receptor Repertoire

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The naive and memory subsets of CD8⁺ and CD4⁺ T cells were isolated from cryopreserved cells using Moflo Astrios EQ (Beckman Coulter), and the dead cells and surface markers were stained with anti-CD4-PE (RPA-T4), anti-CD8-Alexa Fluor 700 (RPA-T8), anti-CD3-BV510 (HIT3a; all from BD Biosciences), anti-CD45RA-APC (HI100; Thermo Fisher), and anti-CCR7-Percp-Cy5.5 (G043H7, BioLegend). We extracted RNA from isolated cells using an RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. CDR3 regions of the TCR chain were amplified using RNA as a template, with primer sets carrying a specific barcode (iRepertoire). The cDNA amplicons were sequenced using HiSeq 4000 (Illumina), as previously described.²⁴ (link) iRepertoire analyzed CDR3 sequences²⁴ (link) and provided raw data regarding variable (V), diversity (D), and joining (J) segment usage and CDR3 length. We measured TCR repertoire diversity using the following indices:

Inverse Simpson index (1 / λ) = 1 / \sum_{i = 1}^{R} p_{i}^{2}

{Shannon index (H}^{'}) = - p_{i} \sum_{i = 1}^{R} \ln p_{i}

Normalized Shannon index = \frac{S h a n n o n i n d e x (H ´)}{\log (t h e n u m b e r o f t o t a l p r o d u c t i v e n t s e q u e n c e s)}

U / T index = \frac{the number of productive unique nt sequences}{the number of total productive nt sequences}

Kim S., Lee S.W., Koh J.Y., Choi D., Heo M., Chung J.Y., Lee B.H., Yang S.H., Sung Y.C., Lee H., Shin E.C, & Park S.H. (2022). A single administration of hIL-7-hyFc induces long-lasting T-cell expansion with maintained effector functions. Blood Advances, 6(23), 6093-6107.

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Profiling Immune Cell Subsets by Flow Cytometry

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The following monoclonal antibodies were used for cell staining: anti-CD3 (FITC; BD Biosciences, San Jose, CA, USA), anti-CD8 (APC; eBioscience, San Diego, CA, USA), anti-CD45RA (PE; BD Biosciences), anti-CCR7 (PerCP/Cy 5.5; BioLegend, San Diego, CA, USA), anti-PD-1 (PE/Cy7; BioLegend), and Fixable Viability Dye (APC-Cy7; eBioscience). Both PBMC and decidual cells were stained with anti-CD3, anti-CD8, anti-CD45RA, anti-CCR7, and anti-PD-1 for 20 min on ice and then incubated for 5 min with Fixable Viability Dye to exclude dead cells. After staining, the cells were washed with PBS. Flow cytometric analysis and single cell sorting were performed using a FACSAria II flow cytometer (BD Biosciences). CD3⁺CD8⁺CD45RA⁺CCR7⁺ cells (naive CD8⁺ T cells; CD8⁺ N cells) and CD3⁺CD8⁺CD45RA⁻CCR7⁻ cells (effector memory CD8⁺ T cells; CD8⁺ EM cells) were single cell sorted into 96-well PCR plates (Supplementary Figures 1A,B). PD-1 expression in each cell was analyzed by the index sort method (Supplementary Figure 1C) (29 (link)).

Morita K., Tsuda S., Kobayashi E., Hamana H., Tsuda K., Shima T., Nakashima A., Ushijima A., Kishi H, & Saito S. (2020). Analysis of TCR Repertoire and PD-1 Expression in Decidual and Peripheral CD8+ T Cells Reveals Distinct Immune Mechanisms in Miscarriage and Preeclampsia. Frontiers in Immunology, 11, 1082.

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